High-Throughput CRISPR Vector Construction and Mutant Characterization in Plants

Targeted mutagenesis with CRISPR is quickly becoming a routine practice in plant biology research. With the explosion of resources and protocols that are being generated, it can be difficult for researchers to know the various options, and how to balance the inherent advantages and disadvantages of each approach. Here we present a method to clone new gRNAs using DNA (Gibson) assembly and compare it to other cloning strategies. Using DNA assembly, CRISPR vectors can be constructed individually, or, by combining multiple oligos into a single reaction, libraries of CRISPR vectors can be generated. This library approach is particularly useful when cloning a large number of gRNAs by reducing the amount of hands-on labor required. In addition, CRISPR libraries can be used directly for Agrobacterium-mediated plant transformation. In tomato, transformation with such Agrobacterium libraries allows for the rapid generation of mutants in several genes of interest. There are also a variety of methods available to identify and characterize mutations. Examples and comparisons from simple Sanger sequencing to high-throughput sequencing will be discussed.